Publications by authors named "Edward M Conway"

90 Publications

CD248 enhances tissue factor (TF) procoagulant function, promoting arterial and venous thrombosis in mouse models.

J Thromb Haemost 2021 Apr 8. Epub 2021 Apr 8.

Centre for Blood Research, Canadian Blood Services, Centre for Innovation.

Background: CD248 is a pro-inflammatory, transmembrane glycoprotein expressed by vascular smooth muscle cells (VSMC), monocytes/macrophages and other cells of mesenchymal origin. Its distribution and properties are reminiscent of those of the initiator of coagulation, tissue factor (TF).

Objective: We examined whether CD248 also participates in thrombosis.

Methods: We evaluated the role of CD248 in coagulation using mouse models of vascular injury, and by assessing its functional interaction with the TF-factor VIIa (FVIIa)-FX complex.

Results: The time to ferric chloride-induced occlusion of the carotid artery in CD248 KO mice was significantly longer than in WT mice. In an inferior vena cava (IVC) stenosis model of thrombosis, lack of CD248 conferred relative resistance to thrombus formation when compared to WT mice. Levels of circulating cells and coagulation factors, PT, aPTT and tail bleeding times were similar in both groups. Proximity ligation assays revealed that TF and CD248 are <40 nm apart, suggesting a potential functional relationship. Expression of CD248 by murine and human vascular smooth muscle cells, and by a monocytic cell line, significantly augmented TF-FVIIa-mediated activation of FX, that was not due to differential expression or encryption of TF, altered exposure of phosphatidylserine or differences in TFPI expression. Rather, conformation specific antibodies showed that CD248 induces allosteric changes in the TF-FVIIa-FX complex that facilitates FX activation by TF-FVIIa.

Conclusion: CD248 is a newly uncovered protein partner and potential therapeutic target in the TF-FVIIa-FX macromolecular complex that modulates coagulation.
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http://dx.doi.org/10.1111/jth.15338DOI Listing
April 2021

VEGF-Induced Endothelial Podosomes via ROCK2-Dependent Thrombomodulin Expression Initiate Sprouting Angiogenesis.

Arterioscler Thromb Vasc Biol 2021 Mar 18:ATVBAHA121315931. Epub 2021 Mar 18.

Department of Biochemistry and Molecular Biology, National Cheng Kung University, Tainan, Taiwan (C.-H.K., P.-K.C., G.-Y.S., H.-L.W.).

Objective: VEGF (vascular endothelial growth factor) plays a critical role in physiological and pathological angiogenesis. Endothelial 3D podosomes (3DPs) are a type of F-actin-rich membrane microdomain, predominantly found in endothelial tip cells controlled by VEGF signaling during sprouting angiogenesis, such as occurs in retinal vasculature development. The molecular mechanisms governing 3DP formation have not been completely elucidated. Approach and Results: By using in vitro cell models and in vivo mouse models, we study the role of TM (thrombomodulin) in VEGF-induced endothelial 3DPs. Here, we report that VEGF can induce the expression of TM via ROCK2 (Rho-associated coiled-coil kinase 2). Furthermore, ROCK2 can catalyze the phosphorylated activation of ezrin to promote the association of the cytoplasmic domain of TM with F-actin in 3DPs and thereby promote the formation of 3DPs. We used endothelial cells transfected with different TM mutants as models to verify the role of TM domains in 3DPs and angiogenic activity. TM expression in endothelial cells augments angiogenic activity, a response that is dependent on the interaction of the cytoplasmic tail of TM with ezrin, and the integrity of the lectin-like domain of TM. Thus, as compared with wild-type counterparts, mice lacking the lectin-like domain of TM exhibit reduced neovascularization of granulation tissues during cutaneous wound healing and less retinal neovascularization in a model of oxygen-induced retinopathy.

Conclusions: VEGF-ROCK2-ezrin-TM-F-actin axis promotes the formation of the lipid raft membrane-associated complex configuration, 3DP, which plays a critical role in mediating tube formation and cell migration of endothelial cells in sprouting angiogenesis.
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http://dx.doi.org/10.1161/ATVBAHA.121.315931DOI Listing
March 2021

Releasates of riboflavin/UV-treated platelets: Microvesicles suppress cytokine-mediated endothelial cell migration/proliferation.

Transfusion 2021 Feb 24. Epub 2021 Feb 24.

Centre for Innovation, Canadian Blood Services, University of British Columbia, Vancouver, British Columbia, Canada.

Background: Accelerated development of the platelet (PLT) storage lesion upon pathogen inactivation (PI) is associated with the release of proteins from granules and platelet microvesicles (PMVs). Whether PI treatments alter the interaction between PLT factors and the vessel endothelium is of interest in understanding the risk profile of these technologies.

Study Design And Methods: In a pool-and-split study, one platelet concentrate (PC) was treated with riboflavin/UV (RF/UV) light, while the other one was kept as an untreated control. Releasates and PMV-depleted releasates were prepared by differential centrifugation steps on days 0, 1, 5, and 7 of storage. Cytokine/chemokine release following PI treatment was analyzed by an antibody array, and results were verified by the enzyme-linked immunosorbent assay. PMVs were enumerated by CD41 labeling and flow cytometry. Wound scratch assays were performed using cultured Ea.hy926 cells exposed to the differently prepared releasates. Effects of releasates on the phosphorylation levels of kinases ERK and p38 expressed by endothelial cells were analyzed by immunoblot.

Results: Cytokine/chemokine assays identified a 2-fold increase in epidermal growth factor released from PCs treated with RF/UV light compared with control. PMV count increased ~100-fold following PI treatment. Unmodified releasates and PMV-depleted releasates displayed different contributions to the kinetics of endothelial cell wound closure. This observation was associated with an increased ERK versus unaltered p38 activation in the endothelial cells.

Conclusion: This study identified an inhibitory impact of PMVs on endothelial cell migration/proliferation upon stimulation by released cytokines and PMVs from PLTs treated with RF/UV light for endothelial cell wound closure.
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http://dx.doi.org/10.1111/trf.16337DOI Listing
February 2021

The association of ABO blood group with indices of disease severity and multiorgan dysfunction in COVID-19.

Blood Adv 2020 10;4(20):4981-4989

Division of Critical Care Medicine, Department of Medicine, University of British Columbia, Vancouver, BC, Canada.

Studies on severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) suggest a protective effect of anti-A antibodies against viral cell entry that may hold relevance for SARS-CoV-2 infection. Therefore, we aimed to determine whether ABO blood groups are associated with different severities of COVID-19. We conducted a multicenter retrospective analysis and nested prospective observational substudy of critically ill patients with COVID-19. We collected data pertaining to age, sex, comorbidities, dates of symptom onset, hospital admission, intensive care unit (ICU) admission, mechanical ventilation, continuous renal replacement therapy (CRRT), standard laboratory parameters, and serum inflammatory cytokines. National (N = 398 671; P = .38) and provincial (n = 62 246; P = .60) ABO blood group distributions did not differ from our cohort (n = 95). A higher proportion of COVID-19 patients with blood group A or AB required mechanical ventilation (P = .02) and CRRT (P = .004) and had a longer ICU stay (P = .03) compared with patients with blood group O or B. Blood group A or AB also had an increased probability of requiring mechanical ventilation and CRRT after adjusting for age, sex, and presence of ≥1 comorbidity. Inflammatory cytokines did not differ between patients with blood group A or AB (n = 11) vs O or B (n = 14; P > .10 for all cytokines). Collectively, our data indicate that critically ill COVID-19 patients with blood group A or AB are at increased risk for requiring mechanical ventilation, CRRT, and prolonged ICU admission compared with patients with blood group O or B. Further work is needed to understand the underlying mechanisms.
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http://dx.doi.org/10.1182/bloodadvances.2020002623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594392PMC
October 2020

Is the COVID-19 thrombotic catastrophe complement-connected?

J Thromb Haemost 2020 11 18;18(11):2812-2822. Epub 2020 Sep 18.

Centre for Blood Research, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada.

In December 2019, the world was introduced to a new betacoronavirus, referred to as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for its propensity to cause rapidly progressive lung damage, resulting in high death rates. As fast as the virus spread, it became evident that the novel coronavirus causes a multisystem disease (COVID-19) that may involve multiple organs and has a high risk of thrombosis associated with striking elevations in pro-inflammatory cytokines, D-dimer, and fibrinogen, but without disseminated intravascular coagulation. Postmortem studies have confirmed the high incidence of venous thromboembolism, but also notably revealed diffuse microvascular thrombi with endothelial swelling, consistent with a thrombotic microangiopathy, and inter-alveolar endothelial deposits of complement activation fragments. The clinicopathologic presentation of COVID-19 thus parallels that of other thrombotic diseases, such as atypical hemolytic uremic syndrome (aHUS), that are caused by dysregulation of the complement system. This raises the specter that many of the thrombotic complications arising from SARS-CoV-2 infections may be triggered and/or exacerbated by excess complement activation. This is of major potential clinical relevance, as currently available anti-complement therapies that are highly effective in protecting against thrombosis in aHUS, could be efficacious in COVID-19. In this review, we provide mounting evidence for complement participating in the pathophysiology underlying the thrombotic diathesis associated with pathogenic coronaviruses, including SARS-CoV-2. Based on current knowledge of complement, coagulation and the virus, we suggest lines of study to identify novel therapeutic targets and the rationale for clinical trials with currently available anti-complement agents for COVID-19.
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http://dx.doi.org/10.1111/jth.15050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7436532PMC
November 2020

Sustained depletion of FXIII-A by inducing acquired FXIII-B deficiency.

Blood 2020 12;136(25):2946-2954

Michael Smith Laboratories.

The activated form of coagulation factor XIII (FXIII-A2B2), FXIII-A*, is a hemostatic enzyme essential for inhibiting fibrinolysis by irreversibly crosslinking fibrin and antifibrinolytic proteins. Despite its importance, there are no modulatory therapeutics. Guided by the observation that humans deficient in FXIII-B have reduced FXIII-A without severe bleeding, we hypothesized that a suitable small interfering RNA (siRNA) targeting hepatic FXIII-B could safely decrease FXIII-A. Here we show that knockdown of FXIII-B with siRNA in mice and rabbits using lipid nanoparticles resulted in a sustained and controlled decrease in FXIII-A. The concentration of FXIII-A in plasma was reduced by 90% for weeks after a single injection and for more than 5 months with repeated injections, whereas the concentration of FXIII-A in platelets was unchanged. Ex vivo, crosslinking of α2-antiplasmin and fibrin was impaired and fibrinolysis was enhanced. In vivo, reperfusion of carotid artery thrombotic occlusion was also enhanced. Re-bleeding events were increased after challenge, but blood loss was not significantly increased. This approach, which mimics congenital FXIII-B deficiency, provides a potential pharmacologic and experimental tool to modulate FXIII-A2B2 activity.
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http://dx.doi.org/10.1182/blood.2020004976DOI Listing
December 2020

Thrombomodulin Functional Domains Support Osteoblast Differentiation and Bone Healing in Diabetes in Mice.

J Bone Miner Res 2020 09 12;35(9):1812-1823. Epub 2020 May 12.

Orthopaedic Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan.

Thrombomodulin (TM) is a transmembrane glycoprotein that contains five functional domains. Soluble TM (sTM), comprising extracellular domains TMD1 (lectin-like), TMD2 (epidermal growth factor [EGF]-like repeat containing), and TMD3 (serine-threonine rich), can be shed from cells by the intramembrane protease rhomboid-like-2 (RHBDL2). TM is expressed by osteoblasts, yet its role there has not been determined. Herein we aimed to investigate the properties of TM and its domains in osteoblast function and bone repair following injury in diabetes. In response to a scratch injury of cultured osteoblast-like MG63 cells, expression of TM and RHBDL2 was enhanced, with increased release of sTM. Conditioned media from the injured cells promoted osteoblast migration, an effect that was lacking with conditioned media from MG63 cells in which TM was silenced by shRNA. Exogenous recombinant TMD1 had no effect on osteoblast activities or on bone repair in vivo. However, TM domains 2 and 3 (TMD2/3), induced MG63 cell migration, proliferation and mineralization in vitro, and when locally administered in mice, improved in vivo healing of injured calvarium. This beneficial effect of TMD2/3, mediated via fibroblast growth factor receptor (FGFR)/ERK signaling pathways, was also observed in vitro under high glucose conditions where endogenous TM expression was reduced, and in vivo in diabetic mice following tibia fracture or calvarium injury, where the osteoblastic response and healing were otherwise dampened. Taken together, osteoblast TM participates in bone healing, and recombinant TMD2/3 holds promise as a novel therapy for diabetic bone defect healing. © 2020 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.4036DOI Listing
September 2020

Platelets and Complement Cross-Talk in Early Atherogenesis.

Front Cardiovasc Med 2019 6;6:131. Epub 2019 Sep 6.

Centre for Blood Research, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada.

Atherosclerosis remains a ubiquitous and serious threat to human health. The initial formation of the atherosclerotic lesion (atheroma) is driven by pro-inflammatory signaling involving monocytes and vascular endothelial cells; later stages of the disease involve rupture of well-established atherosclerotic plaques, thrombosis, and blood vessel occlusion. While the central role of platelets in thrombosis is undisputed, platelets exhibit pro-inflammatory activities, and contribute to early-stage atheroma formation. Platelets also engage components of the complement system, an essential element of innate immunity that contributes to vascular inflammation. Here we provide an overview of the complex interplay between platelets and the complement system, with a focus on how the crosstalk between them may impact on the initiation of atheroma formation.
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http://dx.doi.org/10.3389/fcvm.2019.00131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742699PMC
September 2019

Thrombin: Coagulation's master regulator of innate immunity.

Authors:
Edward M Conway

J Thromb Haemost 2019 11 20;17(11):1785-1789. Epub 2019 Aug 20.

Centre for Blood Research, Life Sciences Institute, Department of Medicine, University of British Columbia, Vancouver, BC, Canada.

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http://dx.doi.org/10.1111/jth.14586DOI Listing
November 2019

Specific loss of adipocyte CD248 improves metabolic health via reduced white adipose tissue hypoxia, fibrosis and inflammation.

EBioMedicine 2019 Jun 17;44:489-501. Epub 2019 Jun 17.

Centre for Blood Research, Life Sciences Institute, Department of Medicine, University of British Columbia, Vancouver, Canada. Electronic address:

Background: A positive energy balance promotes white adipose tissue (WAT) expansion which is characterized by activation of a repertoire of events including hypoxia, inflammation and extracellular matrix remodelling. The transmembrane glycoprotein CD248 has been implicated in all these processes in different malignant and inflammatory diseases but its potential impact in WAT and metabolic disease has not been explored.

Methods: The role of CD248 in adipocyte function and glucose metabolism was evaluated by omics analyses in human WAT, gene knockdowns in human in vitro differentiated adipocytes and by adipocyte-specific and inducible Cd248 gene knockout studies in mice.

Findings: CD248 is upregulated in white but not brown adipose tissue of obese and insulin-resistant individuals. Gene ontology analyses showed that CD248 expression associated positively with pro-inflammatory/pro-fibrotic pathways. By combining data from several human cohorts with gene knockdown experiments in human adipocytes, our results indicate that CD248 acts as a microenvironmental sensor which mediates part of the adipose tissue response to hypoxia and is specifically perturbed in white adipocytes in the obese state. Adipocyte-specific and inducible Cd248 knockouts in mice, both before and after diet-induced obesity and insulin resistance/glucose intolerance, resulted in increased microvascular density as well as attenuated hypoxia, inflammation and fibrosis without affecting fat cell volume. This was accompanied by significant improvements in insulin sensitivity and glucose tolerance.

Interpretation: CD248 exerts detrimental effects on WAT phenotype and systemic glucose homeostasis which may be reversed by suppression of adipocyte CD248. Therefore, CD248 may constitute a target to treat obesity-associated co-morbidities.
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http://dx.doi.org/10.1016/j.ebiom.2019.05.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606747PMC
June 2019

Polyphosphates and Complement Activation.

Authors:
Edward M Conway

Front Med (Lausanne) 2019 4;6:67. Epub 2019 Apr 4.

Division of Hematology, Department of Medicine, Faculty of Medicine, Centre for Blood Research, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada.

To sustain life in environments that are fraught with risks of life-threatening injury, organisms have developed innate protective strategies such that the response to wounds is rapid and localized, with the simultaneous recruitment of molecular, biochemical, and cellular pathways that limit bleeding and eliminate pathogens and damaged host cells, while promoting effective healing. These pathways are both coordinated and tightly regulated, as their over- or under-activation may lead to inadequate healing, disease, and/or demise of the host. Recent advances in our understanding of coagulation and complement, a key component of innate immunity, have revealed an intriguing linkage of the two systems. Cell-secreted polyphosphate promotes coagulation, while dampening complement activation, discoveries that are providing insights into disease mechanisms and suggesting novel therapeutic strategies.
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http://dx.doi.org/10.3389/fmed.2019.00067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458250PMC
April 2019

Advances in Clinical and Basic Science of Coagulation: Illustrated abstracts of the 9th Chapel Hill Symposium on Hemostasis.

Res Pract Thromb Haemost 2018 Jul 12;2(3):407-428. Epub 2018 Apr 12.

Blood Research Institute Blood Center of Wisconsin Milwaukee WI USA.

This 9th Symposium on Hemostasis is an international scientific meeting held biannually in Chapel Hill, North Carolina. The meeting is in large measure the result of the close friendship between the late Dr. Harold R. Roberts of UNC Chapel Hill and Dr. Ulla Hedner of Novo Nordisk. When Novo Nordisk was developing the hemophilia therapy that would become NovoSeven, they sponsored a series of meetings to understand the basic biology and clinical applications of factor VIIa. The first meeting in Chapel Hill was held April 4-6, 2002 with Dr. Roberts as the organizer. Over the years, the conference emphasis has expanded from discussions of factor VIIa and tissue factor to additional topics in hemostasis and thrombosis. This year's meeting includes presentations by internationally renowned speakers that discuss the state-of-the-art on an array of important topics, including von Willebrand factor, engineering advances, coagulation and disease, tissue factor biology, therapeutic advances, and basic clotting factor biology. Included in this review article are illustrated abstracts provided by our speakers, which highlight the main conclusions of each invited talk. This will be the first meeting without Dr. Roberts in attendance, yet his commitment to excellent science and his focus on turning science to patient care are pervasively reflected in the presentations by our speakers.
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http://dx.doi.org/10.1002/rth2.12095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6046595PMC
July 2018

Exploring traditional and nontraditional roles for thrombomodulin.

Blood 2018 07 4;132(2):148-158. Epub 2018 Jun 4.

Centre for Blood Research, Division of Hematology-Oncology, Department of Medicine, University of British Columbia, Vancouver, BC, Canada.

Thrombomodulin (TM) is an integral component of a multimolecular system, localized primarily to the vascular endothelium, that integrates crucial biological processes and biochemical pathways, including those related to coagulation, innate immunity, inflammation, and cell proliferation. These are designed to protect the host from injury and promote healing. The "traditional" role of TM in hemostasis was determined with its discovery in the 1980s as a ligand for thrombin and a critical cofactor for the major natural anticoagulant protein C system and subsequently for thrombin-mediated activation of the thrombin activatable fibrinolysis inhibitor (also known as procarboxypeptidase B2). Studies in the past 2 decades are redefining TM as a molecule with many properties, exhibited via its multiple domains, through its interacting partners, complex regulated expression, and synthesis by cells other than the endothelium. In this report, we review some of the recently reported diverse properties of TM and how these may impact on our understanding of the pathogenesis of several diseases.
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http://dx.doi.org/10.1182/blood-2017-12-768994DOI Listing
July 2018

Complement-coagulation connections.

Authors:
Edward M Conway

Blood Coagul Fibrinolysis 2018 Apr;29(3):243-251

Centre for Blood Research, Division of Hematology-Oncology, Department of Medicine, University of British Columbia, Vancouver, Canada.

: Complement and coagulation are evolutionarily related proteolytic cascades in the blood that are critical for effecting an appropriate innate response to injury that limits bleeding and infection, while promoting healing. Although often viewed as distinct, it has long been recognized that cross-talk likely exists between these pathways. Only recently have molecular links been established. These are providing insights that are revealing opportunities for the development of novel therapeutic strategies to better treat a wide range of thrombotic, inflammatory, immune, infectious, and malignant diseases. In this brief review, the complex relationship between complement and coagulation is highlighted, underlining some of the newly uncovered interactions, in the hopes of stimulating innovative research that will yield improvements in patient outcomes.
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http://dx.doi.org/10.1097/MBC.0000000000000720DOI Listing
April 2018

The Structural Basis for Complement Inhibition by Gigastasin, a Protease Inhibitor from the Giant Amazon Leech.

J Immunol 2017 12 23;199(11):3883-3891. Epub 2017 Oct 23.

Department of Biochemistry and Genetics and Australian Research Council Centre of Excellence in Advanced Molecular Imaging, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia;

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway.
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http://dx.doi.org/10.4049/jimmunol.1700158DOI Listing
December 2017

A novel 2-stage approach that detects complement activation in patients with antiphospholipid antibody syndrome.

Thromb Res 2017 Aug 9;156:119-125. Epub 2017 Jun 9.

Centre for Blood Research, Department of Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Electronic address:

Introduction: The antiphospholipid syndrome (APS) is marked by autoantibodies that recognize anionic phospholipids in a cofactor-dependent manner. A role for complement has been implicated in the pathophysiology, however, elevations of complement activation markers have not been consistently demonstrated in clinical studies. We therefore designed a proof-of-principle study to determine whether complement activation might be detectable in APS by first exposing plasmas to phospholipid vesicles.

Methods: We examined complement activation markers in patients with APS, non-APS thrombosis, systemic lupus erythematosus, cancer, patients with antiphospholipid antibodies without thrombosis (APL) and healthy controls. Direct measurements of plasma C5a and sC5b-9 levels were compared to levels that were generated in normal serum by phospholipid vesicles that had been pre-incubated with the same plasmas. We then determined the effects of the C5 inhibitor, eculizumab, examined the complement pathways involved, and determined whether the effects could be reproduced with purified IgGs and β2-glycoprotein I (β2GPI).

Results: Plasma levels of C5a and sC5b-9 were higher, but not significantly increased in APS patients compared to healthy controls. In contrast, phospholipid vesicles pre-incubated with APS plasmas generated significantly higher levels than healthy controls and the other groups, except for APL patients. Complement activation was abrogated by addition of eculizumab. The results with substrate sera indicated that the alternative and classical/lectin pathways were involved. The results were reproducible with purified IgGs and β2GPI.

Conclusion: This proof-of-principle study confirms a role for complement in APS and opens the possibility of monitoring complement activation by including phospholipid vesicles in assay systems.
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http://dx.doi.org/10.1016/j.thromres.2017.06.014DOI Listing
August 2017

Diagnosis of Western Red Cedar Asthma Using a Blood-based Gene Expression Biomarker Panel.

Am J Respir Crit Care Med 2017 12;196(12):1615-1617

1 University of British Columbia Vancouver, British Columbia, Canada and.

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http://dx.doi.org/10.1164/rccm.201608-1740LEDOI Listing
December 2017

Alteration of blood clotting and lung damage by protamine are avoided using the heparin and polyphosphate inhibitor UHRA.

Blood 2017 03 29;129(10):1368-1379. Epub 2016 Dec 29.

Centre for Blood Research.

Anticoagulant therapy-associated bleeding and pathological thrombosis pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids. The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described cationic inhibitors of polyP and extracellular nucleic acids exhibit both nonspecific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation used to counteract heparin-associated bleeding in surgical settings, protamine, exhibits adverse effects. To address these clinical shortcomings, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. Sharply contrasting protamine, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization during clot formation, or abrogate plasma clotting. Using scanning electron microscopy, confocal microscopy, and clot lysis assays, we confirm that UHRA does not incorporate into clots, and that clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury seen with protamine. The data presented here illustrate that UHRA could be safely used as an antidote during adverse therapeutic modulation of hemostasis.
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http://dx.doi.org/10.1182/blood-2016-10-747915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345737PMC
March 2017

Polyphosphate/platelet factor 4 complexes can mediate heparin-independent platelet activation in heparin-induced thrombocytopenia.

Blood Adv 2016 Nov 22;1(1):62-74. Epub 2016 Nov 22.

Faculty of Medicine and Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada.

Heparin-induced thrombocytopenia (HIT) is a thrombotic disorder initiated by antibodies to complexes between platelet factor 4 (PF4) and heparin. The risk of recurrent thromboembolism persists after heparin is cleared and platelet activation leading to release of PF4 has dissipated. We asked whether antigenic complexes between polyphosphates and PF4 released from activated platelets might intensify or sustain the prothrombotic phenotype of HIT. PF4 forms stable, ultralarge complexes with polyphosphates of various sizes, including those released from platelets, which are recognized by the HIT-like monoclonal KKO, an immunoglobulin G2bκ monoclonal heparin/PF4 binding antibody, and by human HIT antibodies. KKO helps to protect PF4/polyphosphate complexes from degradation by phosphatases. Complement is activated when HIT antibodies bind to PF4/polyphosphate complexes and PF4 reverses the inhibition of complement by polyphosphates. Polyphosphates and PF4 are stored primarily in separate granules in resting platelets, but they colocalize when the cells are activated. Platelets activated by subaggregating doses of thrombin receptor activating peptide release polyphosphates and PF4, which form antigenic complexes that allow KKO to further activate platelets in the absence of heparin and exogenous PF4. These studies suggest that thrombin- or immune complex-mediated release of endogenous antigenic PF4/polyphosphate complexes from platelets may augment the prothrombotic risk of HIT and perpetuate the risk of thrombosis after heparin has been discontinued.
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http://dx.doi.org/10.1182/bloodadvances.2016000877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744055PMC
November 2016

Polyphosphate is a novel cofactor for regulation of complement by a serpin, C1 inhibitor.

Blood 2016 09 23;128(13):1766-76. Epub 2016 Jun 23.

Centre for Blood Research, Department of Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada;

The complement system plays a key role in innate immunity, inflammation, and coagulation. The system is delicately balanced by negative regulatory mechanisms that modulate the host response to pathogen invasion and injury. The serpin, C1-esterase inhibitor (C1-INH), is the only known plasma inhibitor of C1s, the initiating serine protease of the classical pathway of complement. Like other serpin-protease partners, C1-INH interaction with C1s is accelerated by polyanions such as heparin. Polyphosphate (polyP) is a naturally occurring polyanion with effects on coagulation and complement. We recently found that polyP binds to C1-INH, prompting us to consider whether polyP acts as a cofactor for C1-INH interactions with its target proteases. We show that polyP dampens C1s-mediated activation of the classical pathway in a polymer length- and concentration-dependent manner by accelerating C1-INH neutralization of C1s cleavage of C4 and C2. PolyP significantly increases the rate of interaction between C1s and C1-INH, to an extent comparable to heparin, with an exosite on the serine protease domain of the enzyme playing a major role in this interaction. In a serum-based cell culture system, polyP significantly suppressed C4d deposition on endothelial cells, generated via the classical and lectin pathways. Moreover, polyP and C1-INH colocalize in activated platelets, suggesting that their interactions are physiologically relevant. In summary, like heparin, polyP is a naturally occurring cofactor for the C1s:C1-INH interaction and thus an important regulator of complement activation. The findings may provide novel insights into mechanisms underlying inflammatory diseases and the development of new therapies.
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http://dx.doi.org/10.1182/blood-2016-02-699561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043130PMC
September 2016

Sweeteners for factor H.

Authors:
Edward M Conway

Blood 2016 06;127(22):2656-8

UNIVERSITY OF BRITISH COLUMBIA CENTRE FOR BLOOD RESEARCH.

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http://dx.doi.org/10.1182/blood-2016-04-708172DOI Listing
June 2016

Hepatocellular carcinoma repression by TNFα-mediated synergistic lethal effect of mitosis defect-induced senescence and cell death sensitization.

Hepatology 2016 10 11;64(4):1105-20. Epub 2016 Jun 11.

State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

Unlabelled: Hepatocellular carcinoma (HCC) is a cancer lacking effective therapies. Several measures have been proposed to treat HCCs, such as senescence induction, mitotic inhibition, and cell death promotion. However, data from other cancers suggest that single use of these approaches may not be effective. Here, by genetic targeting of Survivin, an inhibitor of apoptosis protein (IAP) that plays dual roles in mitosis and cell survival, we identified a tumor necrosis factor alpha (TNFα)-mediated synergistic lethal effect between senescence and apoptosis sensitization in malignant HCCs. Survivin deficiency results in mitosis defect-associated senescence in HCC cells, which triggers local inflammation and increased TNFα. Survivin inactivation also sensitizes HCC cells to TNFα-triggered cell death, which leads to marked HCC regression. Based on these findings, we designed a combination treatment using mitosis inhibitor and proapoptosis compounds. This treatment recapitulates the therapeutic effect of Survivin deletion and effectively eliminates HCCs, thus representing a potential strategy for HCC therapy.

Conclusion: Survivin ablation dramatically suppresses human and mouse HCCs by triggering senescence-associated TNFα and sensitizing HCC cells to TNFα-induced cell death. Combined use of mitotic inhibitor and second mitochondrial-derived activator of caspases mimetic can induce senescence-associated TNFα and enhance TNFα-induced cell death and synergistically eliminate HCC. (Hepatology 2016;64:1105-1120).
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http://dx.doi.org/10.1002/hep.28637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5089570PMC
October 2016

Cross Talk Pathways Between Coagulation and Inflammation.

Circ Res 2016 Apr;118(9):1392-408

From the Department of Haematology, UCL Cancer Institute, University College London, London, United Kingdom (J.H.F.); Katharine Dormandy Haemophilia Centre and Thrombosis Unit, Royal Free NHS Trust, London, United Kingdom (J.H.F.); and Centre for Blood Research, Department of Medicine, University of British Columbia, Vancouver, Canada (E.M.C.).

Anatomic pathology studies performed over 150 years ago revealed that excessive activation of coagulation occurs in the setting of inflammation. However, it has taken over a century since these seminal observations were made to delineate the molecular mechanisms by which these systems interact and the extent to which they participate in the pathogenesis of multiple diseases. There is, in fact, extensive cross talk between coagulation and inflammation, whereby activation of one system may amplify activation of the other, a situation that, if unopposed, may result in tissue damage or even multiorgan failure. Characterizing the common triggers and pathways are key for the strategic design of effective therapeutic interventions. In this review, we highlight some of the key molecular interactions, some of which are already showing promise as therapeutic targets for inflammatory and thrombotic disorders.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.306853DOI Listing
April 2016

Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin.

EBioMedicine 2016 Mar 6;5:175-82. Epub 2016 Feb 6.

Centre for Blood Research, Department of Medicine, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, LSC4306, Vancouver V6T 1Z3, Canada.

Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin-antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution.
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http://dx.doi.org/10.1016/j.ebiom.2016.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816834PMC
March 2016

A Nuclear Attack on Thrombosis and Inflammation.

Authors:
Edward M Conway

Arterioscler Thromb Vasc Biol 2016 Feb;36(2):221-3

From the Department of Medicine, Centre for Blood Research, University of British Columbia, Vancouver, Canada.

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http://dx.doi.org/10.1161/ATVBAHA.115.306979DOI Listing
February 2016

Essential Role for Survivin in the Proliferative Expansion of Progenitor and Mature B Cells.

J Immunol 2016 Mar 25;196(5):2195-204. Epub 2016 Jan 25.

Tumor Microenvironment and Cancer Immunology Program, National Cancer Institute-Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037; and

Survivin is a member of the inhibitor of apoptosis family of proteins and a biomarker of poor prognosis in aggressive B cell non-Hodgkin's lymphoma. In addition to its role in inhibition of apoptosis, survivin also regulates mitosis. In this article, we show that deletion of survivin during early B cell development results in a complete block at the cycling pre-B stage. In the periphery, B cell homeostasis is not affected, but survivin-deficient B cells are unable to mount humoral responses. Correspondingly, we show that survivin is required for cell division in response to mitogenic stimulation. Thus, survivin is essential for proliferation of B cell progenitors and activated mature B cells, but is dispensable for B cell survival. Moreover, a small-molecule inhibitor of survivin strongly impaired the growth of representative B lymphoma lines in vitro, supporting the validity of survivin as an attractive therapeutic target for high-grade B cell non-Hodgkin's lymphoma.
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http://dx.doi.org/10.4049/jimmunol.1501690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4761464PMC
March 2016

The lectin like domain of thrombomodulin is involved in the defence against pyelonephritis.

Thromb Res 2015 Dec 10;136(6):1325-31. Epub 2015 Nov 10.

Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Pyelonephritis, a common complication of urinary tract infections, is frequently associated with kidney scarring and may lead to end-stage renal disease. During bacterial infections inflammatory and coagulation pathways and their mutual interaction are playing pivotal roles in the host response. Given that thrombomodulin (TM) is crucially involved in the interplay between coagulation and inflammation, we aimed to investigate the roles of its EGF and lectin-like domains in inflammation during acute pyelonephritis. Indeed, the EGF-like and the lectin-like domains of TM, are especially known to orchestrate inflammation and coagulation in different ways. Acute pyelonephritis was induced by intravesical inoculation of 1 × 10(8) CFU of uropathogenic Escherichia coli in two strains of TM transgenic mice. TM(pro/pro) mice carry a mutation in the EGF-like domain making them unable to activate protein C, an anticoagulant and anti-inflammatory protein. TM(LeD/LeD) mice lack the lectin-like domain of TM, which is critical for its anti-inflammatory and cytoprotective properties. Mice were sacrificed 24 and 48 h after inoculation. Bacterial loads, the immune response and the activation of coagulation were evaluated in the kidney and the bladder. TM(LeD/LeD) mice showed elevated bacterial load in bladder and kidneys compared to WT mice, whereas TM(pro/pro) had similar bacterial load as WT mice. TM(LeD/LeD) mice displayed a reduced local production of pro-inflammatory cytokines and neutrophil renal infiltration. Activation of coagulation was comparable in TM(LeD/LeD) and WT mice. From these data, we conclude that the lectin-like domain of thrombomodulin is critically involved in host defence against E. coli induced acute pyelonephritis.
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http://dx.doi.org/10.1016/j.thromres.2015.11.004DOI Listing
December 2015

HUS and the case for complement.

Authors:
Edward M Conway

Blood 2015 Oct 22;126(18):2085-90. Epub 2015 Sep 22.

Centre for Blood Research, Life Sciences Institute, Department of Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada.

Hemolytic-uremic syndrome (HUS) is a thrombotic microangiopathy that is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Excess complement activation underlies atypical HUS and is evident in Shiga toxin-induced HUS (STEC-HUS). This Spotlight focuses on new knowledge of the role of Escherichia coli-derived toxins and polyphosphate in modulating complement and coagulation, and how they affect disease progression and response to treatment. Such new insights may impact on current and future choices of therapies for STEC-HUS.
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http://dx.doi.org/10.1182/blood-2015-03-569277DOI Listing
October 2015

Hi-Fi SELEX: A High-Fidelity Digital-PCR Based Therapeutic Aptamer Discovery Platform.

Biotechnol Bioeng 2015 Aug;112(8):1506-22

Current technologies for aptamer discovery typically leverage the systematic evolution of ligands by exponential enrichment (SELEX) concept by recursively panning semi-combinatorial ssDNA or RNA libraries against a molecular target. The expectation is that this iterative selection process will be sufficiently stringent to identify a candidate pool of specific high-affinity aptamers. However, failure of this process to yield promising aptamers is common, due in part to (i) limitations in library designs, (ii) retention of non-specific aptamers during screening rounds, (iii) excessive accumulation of amplification artifacts, and (iv) the use of screening criteria (binding affinity) that does not reflect therapeutic activity. We report a new selection platform, High-Fidelity (Hi-Fi) SELEX, that introduces fixed-region blocking elements to safeguard the functional diversity of the library. The chemistry of the target-display surface and the composition of the equilibration solvent are engineered to strongly inhibit non-specific retention of aptamers. Partition efficiencies approaching 10(6) are thereby realized. Retained members are amplified in Hi-Fi SELEX by digital PCR in a manner that ensures both elimination of amplification artifacts and stoichiometric conversion of amplicons into the single-stranded library required for the next selection round. Improvements to aptamer selections are first demonstrated using human α-thrombin as the target. Three clinical targets (human factors IXa, X, and D) are then subjected to Hi-Fi SELEX. For each, rapid enrichment of ssDNA aptamers offering an order-nM mean equilibrium dissociation constant (Kd) is achieved within three selection rounds, as quantified by a new label-free qPCR assay reported here. Therapeutic candidates against factor D are identified.
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http://dx.doi.org/10.1002/bit.25581DOI Listing
August 2015

Macrophage matrix metalloproteinase-12 dampens inflammation and neutrophil influx in arthritis.

Cell Rep 2014 Oct 9;9(2):618-32. Epub 2014 Oct 9.

Centre for Blood Research, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Electronic address:

Resolution of inflammation reduces pathological tissue destruction and restores tissue homeostasis. Here, we used a proteomic protease substrate discovery approach, terminal amine isotopic labeling of substrates (TAILS), to analyze the role of the macrophage-specific matrix metalloproteinase-12 (MMP12) in inflammation. In murine peritonitis, MMP12 inactivates antithrombin and activates prothrombin, prolonging the activated partial thromboplastin time. Furthermore, MMP12 inactivates complement C3 to reduce complement activation and inactivates the chemoattractant anaphylatoxins C3a and C5a, whereas iC3b and C3b opsonin cleavage increases phagocytosis. Loss of these anti-inflammatory activities in collagen-induced arthritis in Mmp12(-/-) mice leads to unresolved synovitis and extensive articular inflammation. Deep articular cartilage loss is associated with massive neutrophil infiltration and abnormal DNA neutrophil extracellular traps (NETs). The NETs are rich in fibrin and extracellular actin, which TAILS identified as MMP12 substrates. Thus, macrophage MMP12 in arthritis has multiple protective roles in countering neutrophil infiltration, clearing NETs, and dampening inflammatory pathways to prepare for the resolution of inflammation.
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http://dx.doi.org/10.1016/j.celrep.2014.09.006DOI Listing
October 2014